In both in vivo and in vitro environments, cells are exposed to numerous extracellular stimuli that regulate their function. For instance, cellular events in dermal wound healing are strongly influenced by soluble and immobilized biomolecule cues as well as the mechanical environment. The characterization of how cells interpret and respond to these influential cues delivered both individually and in combination is important in not only informing the construction of environments that allow greater control over cell behavior and wound healing, but also in understanding native physiological phenomena. The goal of the proposed research is to characterize how cells interpret, integrate, and respond to simple and complex combinations of microenvironmental cues, with a specific focus on keratinocyte migration and dermal wound healing events. The combination of stimuli that promote accelerated and directed cell migration can be used to inform the design of wound dressing materials that enable faster and more efficient wound closure. This work is intended to not only apply to the clinical problem of wound healing, but also to help scientists and engineers better understand and predict cellular decision-making processes in order to achieve greater control over cell function. The following aims will allow us to characterize and predict how keratinocytes integrate soluble, immobilized, and mechanical cues to make decisions about proliferation, migration, and apoptosis. Specific Aim 1: Characterize the combinatorial effects of soluble EGF, immobilized EGF, and substrate mechanics on keratinocyte signaling and function. In this aim we will quantify the individual and combined effects of soluble epidermal growth factor (EGF), immobilized EGF, and substrate mechanics on cellular behavior to determine how keratinocytes integrate and respond to signals received from these different stimuli, particularly in the context of dermal wound healing events. Specific Aim 2: Characterize the contributions and integration of additive and opposing chemotactic, haptotactic, and durotactic gradient stimuli with respect to regulation of keratinocyte signaling and function. The purpose of this aim is to understand the contributions of spatially-directed soluble, immobilized, and mechanical stimuli with respect to controlling the function of keratinocytes. Specifically, chemotactic, haptotactic, and durotactic gradient stimuli will be combined in both additive and opposing manners in order to examine how cells interpret and respond to these different cues, as well as to determine which cues dominate cell behavior and why. Specific Aim 3: Develop a data-driven network model to interpret and predict multivariate connections in the microenvironmental regulation of keratinocyte signaling and function. A partial least squares regression model will be developed to analyze the data set acquired in Aims 1 and 2, enabling us to not only describe but also predict how keratinocytes integrate numerous combinations of microenvironmental cues. PUBLIC HEALTH RELEVANCE: Cells in the body receive signals from their environment that instruct them on how to function. In this proposal, we aim to better understand how cells integrate and respond to these signals, particularly in the context of dermal wound healing. By studying how these signals stimulate different wound healing events, we will gain information that will help us to design improved wound dressings and treatments.